1. Field of the Invention
The present invention generally relates to amplification methods and more particularly, amplification including midivariant DNA/probe conjugates.
2. Technical Review
Nucleic acid based test assays provide distinct advantages in specificity and sensitivity over conventional immunoassay procedures. The essential feature of nucleic acid based test assays is the hybridization of target specific nucleic acid sequences (probes) to their complementary target nucleic acid sequences in a test sample. A probe's specificity relates to its ability to distinguish between target and non-target nucleic acid sequences. Probe specificity may be absolute (i.e. probe able to distinguish between target nucleic acid sequences and non-target nucleic acid sequences), or it may be functional (i.e. probe able to distinguish between the target nucleic acid sequence and any other nucleic acid sequence normally present in a test sample). A target nucleic acid sequence of a test sample in a test assay refers to a segment of single-stranded polynucleotide having a nucleotide base sequence corresponding to a genetic element whose presence in a test sample is to be determined. The test sample refers to any test sample containing one or more target nucleic acids and which may be in purified or nonpurified form. Sources for test sample may include, but should not be limited to, physiological and nonphysiological DNA or RNA (synthesized or natural), and the like.
It has been noted that the whole repertoire of antigenic determinants of two separate but related organisms may not permit their discrimination by immunoassay, whereas unique genomic sequences can be identified and differentiated by a nucleic acid test assay. See U.S. Pat. No. 4,851,336. Also, in the case of viral targets, i.e. HIV infection, antibodies to viral antigens may not be detectable even though viral sequences have been inserted into the host genome, however, the insertion will provide a potential diagnostic marker if a detection (amplification) methodology of the requisite sensitivity can be devised. The present invention provides amplification methodology(s) of the requisite sensitivity.
It is now realized that the full capability, in terms of sensitivity, of nucleic acid based test assays can only be achieved in conjunction with a method for the amplification of a detectable molecule indicative of the presence of the target nucleic acid sequence in the test sample.
The polymerase chain reaction (PCR) is one type of amplification method where the target sequence is first amplified and then isolated. Since PCR amplification occurs early in the process, sequences other than the target may also amplify. Identification of the actual target requires the analysis of the amplified sequences. See U.S. Pat. Nos. 4,683,195 and 4,683,202.
U.S. Pat. Nos. 4,786,600 and 4,957,858 describe an autocatalytic replication of recombinant RNA by QB replicase, another type of amplification method where the target sequence is first isolated and then amplified. The disadvantage of this method of QB amplification is that probes not hybridized to the target nucleic acid sequence must be made vulnerable to destruction i.e. rendered unreplicable. See "Amplifying Probe Assays with Q-Beta Replicase" Bio/Technology 1989: 7(6), 609-10 (Eng.) and "Q Beta Amplification" J. Clin. Lab. Anal. 4:318 (1990) (letter).